Recent developments in recombinant DNA technology allow one to express genes from higher organisms in bacteria and yeast, which can be grown in large scale fermentations. This technology has spawned a number of efforts to develop commercially viable fermentation processes in which these microorganisms produce proteins such as enzymes or hormones, which must otherwise be isolated from animal or human tissue. In many cases much of the cost of such fermentative processes is in the steps required to recover the desired product in an acceptably pure state. Usually, the microorganisms must be disrupted, and their contents solubilized with denaturants before the desired product can be isolated and purified away from other cell components. Each processing step adds to the final cost of the product.
One way to avoid such a lengthy purification procedure is to arrange for the microorganism to secrete the desired product directly into its growth medium during fermentation. In this way the product can be obtained immediately, in a relatively pure form, simply by removing the producing cells in a single centrifugation or filtration step.
The yeast Saccharomyces cerevisiae has often been put forward as the microorganism of choice for secretory production of protein products. Because it has been used for centuries in the baking and brewing industries, much is known about growing this species on a large scale. Also, it is known to be capable of secreting a significant portion of the protein it produces.
However, previous attempts to use Saccharomyces cerevisiae to produce and secrete protein products from heterologous genes have had mixed success. The secreted yield of protein product was dependent upon both the gene to be expressed and the promoter and signal sequences chosen for its expression (Hitzeman, R. A., Leung, D. W., Perry, L. J., Kohr, W. J., Levine, H. L. and Goeddel, D. V. (1983) Science 219, 620-625; Bitter, G. A., Chen, K. K., Banks, A. R. and Lai, P.-H. (1984) Proc. Natl. Acad. Sci. USA 81, 5330-5334; Brake, A. J., Merryweather, J. P., Coit, D. G., Heberlein, U. A., Masiarz, F. R., Mullenbach, G. T., Urdea, M. S., Valenzuela, P. and Barr, P. J. (1984) Proc. Natl. Acad. Sci. USA 81, 4642-4646; Brake, A. J., Cousens, L. S., Urdea, M. S., Valenzuela, P. D. T. (1984) European Patent Application Publication No. 0 121 884). Although it is usually possible to obtain reasonably good production levels for a particular protein, often only a small fraction of the total amount produced can actually be found free in the medium. Most of the protein remains trapped inside the cell, often in the intracellular vacuole found in this species. In yeast, secretion can be regarded as a branched pathway with some secreted yeast proteins being "secreted" into the vacuole and others being directed across the plasma membrane to the periplasm and beyond (Sheckman, R. and Novick, P., in Strathern, J. N., Jones, E. W. and Broach, J. R. (eds.), Molecular Biology of the Yeast Saccharomyces cerevisiae, Cold Springs Harbor Laboratory, Cold Springs Harbor, New York, 1981, pp. 361-398). Apparently, some protein products of foreign genes are directed into the vacuolar branch of this pathway.
Recent studies by Rothstein, J. H. and Stevens, T. H. (Presentation at the Genetics Society of America Annual Meeting, Aug. 13, 1984) and Emr, S. (Presentation at Yeast Expression Vectors Symposium, Banbury Center, Cold Spring Harbor Laboratory, Jun. 21--24, 1984) have shown that host cell mutations can be isolated that affect the intracellular localization of yeast proteins in the yeast secretion pathway. Both a natural yeast protein (carboxy peptidase Y) and a fusion of two yeast proteins (carboxy peptidase Y and invertase) were redirected from the vacuole to the outside of the cell by host cell mutagenesis and selection. However, the utility of using the mutant strains generated by either of these selection methods for the secretory production of foreign proteins has not been demonstrated. Furthermore, in each case a strong selective pressure for the extracellular secretion of a specific protein was applied to the mutagenized cells. This approach differs greatly from a quantitative screening assay for a nonessential gene product and may have introduced strong bias in the distribution of mutants obtained. Also, neither of these studies were directed at increasing the yield of secreted proteins per se, rather they were designed to isolate mutations to aid in the general study of yeast secretion.
One group has reported an attempt to isolate mutant stains of yeast that oversecrete the killer toxin which is associated with some yeast strains (Bussey, H., Steinmetz O., and Saville, D. (1983) Current Genetics, 7:449-456). However, this group succeeded only in isolating chromosomal mutations that reduced the rate of toxin proteolysis, or reduced the amount bound to the cell wall of producing cells. The actual translocation of this polypeptide in the yeast secretion pathway was unaffected.